The present invention relates to the use of 4-fluorobenzenesulfonyl Chloride for activating hydroxyl groups of polymeric carriers forming biospecific affinity supports. More specifically, the present invention is a method of forming biospecific affinity supports involving the activation of the hydroxyl groups of polymeric carriers with 4-fluorobenzenesulfonyl Chloride for subsequent reaction with biologically active organic ligand having amino or sulfohydryl groups, the biospecific affinity support is useful in a separation procedure.
Recent advances in separation technology allow isolating a specific target population, e.g. cells, proteins, or antibodies, from a heterogeneous solution without the need of tedious and extensive chemical separation techniques. These recent advances involve the use of biospecific affinity supports. Biospecific affinity supports are typically formed from hydroxyl bearing polymeric carriers, in the form of columns, gels or polymeric beads, to which a biologically active organic ligand is chemically bonded. The biologically active organic ligand has selective affinity for bonding to the desired target population, i.e. proteins, enzymes, antibodies and antigens, from solutions.
Biologically active organic ligand are compounds that selectively bind to the desired biologically active target population. One specific example of a biologically active target population is an antigen or enzyme, with the biologically active organic ligand being an antibody selective for the biologically active target population.
Biospecific affinity supports are used in various types of separation techniques. For example, biospecific affinity supports are used in immunologic methods and in affinity chromatography wherein antibodies or antigens are bonded to water insoluble polymeric carriers to function as the biologically active organic ligand.
Various workers have also suggested capturing specific target populations by using filters bearing biologically active organic ligand. These techniques may use filters prepared from fibers, e.g. disclosed in U.S. Pat. No. 3,843,324, issued on Oct. 22, 1974; or columns, e.g. disclosed in U.S. Pat. No. 4,252,653, issued on Feb. 24, 1981; or filter candles, e.g. disclosed in U.S. Pat. No. 4,648,974, issued Mar. 10, 1987.
Another recently developed technique for isolating target populations, i.e. antibodies, selected proteins and cells, from a physiological fluid utilizes paramagnetic beads or particles coated with an biologically active organic ligand selective for the desired target population. Examples of such particles or beads are disclosed in U.S. Pat. Nos. 4,230,685, issued Oct. 28, 1980; 4,554,088, issued Nov. 19, 1985; and 4,628,037, issued Dec. 9, 1986. The use of such particles in the separation of cells is taught in publications, "Removal of Neuroblastoma Cells From Bone Marrow with Monoclonal Antibodies Conjugated to Magnetic Microspheres", by J. G. Treleaven, J. Ugelstad, T. Philips, F. M. Gibson, A. Rembaum, G. D. Caines and J. T. Kemshead, The Lancet, Jan. 14, 1984, pages 70-73, and "Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use", by G. Kvalheim, O. Sorensen, O. Fodstad, S. Funderud, S. Kiesel, B. Dorken, K. Nustad, E. Jakobsen, J. Ugelstad and A. Pihl, Bone Marrow Transplantation, (1988), volume 3, pages 31-41.
There are various known methods for chemically coupling biologically active ligand to hydroxyl bearing polymeric substrates. Some workers demonstrated the use of cyanogen bromide (CNBr) as a compound for activating the hydroxyl groups of hydroxyl bearing polymeric substrates. The biologically active organic ligand is then reacted with the activated hydroxyl groups, see Scouten, et al "Methods in Enzymology 135, 79 (1987). There are disadvantages in using cyanogen bromide for activating hydroxyl groups of polymeric carriers. The chemical linkages formed between the hydroxyl bearing polymeric substrates and biologically active organic ligand are known to be liable. The activated polymeric carrier is not stable, and cyanogen bromide is a noxious, lachrymator and poisonous chemical which requires special handling procedures.
Other workers suggest the use of 2-Fluoro-1-methyl-pyridinium toluene-4-sulfonate (FMP), see U.S. Pat. No. 4,582,875, issued to Ngo on Apr. 15, 1986. A disadvantage associated with FMP as an activating agent is the formation of a positively charged quaternary amine group on the chemically activated polymeric carrier. The presence of the positively charged group makes displacement of this group by similarly charged ligand more difficult and less efficient.
Other workers have activated the hydroxyl groups of hydroxyl bearing polymeric substrates using sulfonyl halogenids (sulfonyl halides), see U.S. Pat. No. 4,415,665, issued to Mosbach et al on Nov. 15, 1983. This patent describes a method utilizing a sulfonyl halogenoid having the formula: EQU Hal--SO.sub.2 --R
wherein R is any organic group suitable for the sulfonyl chloride. Examples of suitable R groups include p-tolyl, p-nitrolphenyl, trifluoroethyl, trifluoromethyl or methyl. While Mosbach et al discloses the use of sulfonyl halogenids in general, the patentees only utilize two specific halogenids, p-toluenesulfonyl and 2,2,2-trifluoroethanesulfonyl chloride. There are distinct disadvantages with each of these halogenids or halides. P-toluenesulfonyl chloride has been demonstrated as being primarily reactive with the secondary, but not the primary hydroxyl groups of the hydroxyl bearing polymeric substrate. This limits the degree of activation obtainable with p-toluenesulfonyl, which limits the amount of ligand that can be attached to the carrier. It has also been demonstrated that reaction between the p-toluenesulfonate groups and the biologically active organic ligand often requires 16 to 24 hours incubation under conditions of Ph 8.0 or higher, which may be damaging to sensitive biologically active organic ligand, such as enzymes or antibodies. Damage to these ligand decreases the usefulness of the ligand coupled carrier.
2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) is toxic, highly reactive and volatile, making use of this material for the activation of the hydroxyl groups of polymeric carriers difficult. Tresyl chloride is typically used in well-ventilated environments. The volatility and high reactivity of tresyl chloride requires that the activation be performed under water-free solvent; dried solvents, rather than commercially available solvents. The use of commercially available solvents lowers the yield of the activation reaction. Unlike the other activation methods, the introduction of the tresyl ester, which results from the reaction of tresyl chloride and the hydroxyl groups, can not be followed with UV spectroscopy. Tresyl chloride is also more expensive than other halides.
The present invention is an improvement of the method disclosed in the Mosbach patent. The applicants' have found that a specific sulfonyl halogenoid, 4-fluorobenzenesulfonyl Chloride, provides unexpected benefits over sulfonyl halogenids in general, and p-toluenesulfonyl and 2,2,2-trifluoroethanesulfonyl chloride in particular.